Systems biology is concerned with elucidating interaction networks that underlie cellular functions, in particular, interactions of proteins. Identification of which proteins interact in, for example, a signaling pathway, provides information that can lead to strategies for controlling such pathways. Thus, knowledge that protein A interacts with protein B opens the possibility of controlling the action of protein B by targeting protein A. Further, understanding the structural features of the interacting proteins as they relate to each other provides information useful in the design of small molecules that might interfere with, or encourage, the interaction, as well as diagnostic tools for understanding mutations in the protein and sequences that may interfere with, or encourage, such interactions.
Multiple approaches have been used to study such interactions, including X-ray crystallography, and nuclear magnetic resonance (NMR) spectroscopy. Other methods used to identify interacting proteins include the yeast-two-hybrid method and in vitro binding assays with purified proteins. In addition, affinity purification of the complexes combined with mass spectrometry (MS) of the complex itself has been used. None of these methods, however, is sufficiently convenient and informative to provide satisfactory data.
Chowdhury, S. M., et al., Anal. Chem. (2006) 78:8183-8193, which is incorporated herein by reference, provide a general description of a particularly useful MS method and describe several crosslinking agents that may be employed. In brief, this method is applied either to a purified complex or to a more crude extract of cellular material and employs crosslinking agents that are able to link amino acid chains that contain functional groups—i.e., amines or sulfhydryl groups—that are reactive with functional groups at each end of a bifunctional linking moiety. Typically, the proximity of these groups either on the same protein chain or on chains of different proteins is less than 100 Å. After the above-mentioned functional groups in sufficient proximity have been chemically crosslinked, the proteins are treated with proteases, typically trypsin. Trypsin reduces the size of the peptides at either end of the crosslinker, which is favorable for subsequent MS analysis. The crosslinkers employed by Chowdhury also contain a chemical moiety separating the two reactive groups that contains a bond that is particularly labile during MS analysis. In addition, the crosslinkers contain a biotin residue that permits affinity purification of the crosslinked and trypsin-treated peptides. The resulting isolated crosslinked peptides are then subjected to MS analysis.
In Chowdhury's method, after biotin affinity enrichment, the majority of the ions detected by MS will be crosslinker-modified peptides, including monolinks, crosslinks, and looplinks. Monolinks usually predominate. Crosslinker-modified peptides are isolated and subjected to collision induced dissociation (CID) conditions that effect cleavage of the crosslinker at the cleavage site. In CID, the isolated peptide ion is collided with inert gas (such as argon), resulting in fragmentation of the peptide at MS labile bond. In the case of an interpeptide crosslink, the CID liberates the two peptides coupled to portions of the crosslinker as well as a reporter molecule derived from the crosslinker. A mass spectrum of the components is obtained. The individual peptide-containing components are then subjected to further fragmentation by CID to obtain characteristic mass spectra, which can be interpreted by database search algorithms to identify the amino acid sequence of the peptides associated with the remains of the crosslinker. Because databases are available to associate sequences with known proteins, identification of the individual peptides associated with the crosslinker is often sufficient to permit identification of the linked proteins. The location of the site of cros slinking is determined directly from the MS fragmentation spectrum of the peptide.
Other crosslinkers used in this general method are described in Lu, Y., et al., Anal. Chem. (2008) 80:9279-9281 and Soderblom, E. J., et al., Rapid Commun. Mass Spectrom. (2007) 21:3395-3408, and those shown in FIG. 1A.